More than 50% of the food used by man is provided by a single group of crop plants, the cereals, of which wheat is the most important species. Biotechnological methods, which provide new and unique opportunities for the genetic improvement of this crop, require efficient plant regeneration in vitro and the recovery of transgenic plants following delivery and integration of foreign genes into regenerable cells (Vasil, I. K. [1988] Bio/Technology 6:397-401).
A transgenic plant can only result from integrative transformation of a totipotent cell or a cell that has a clonal connection to the "germline." A number of methods have been proposed and used for the genetic transformation of plants, including cereal species (Vasil, I. K. [1990] Bio/Technology 8:797; Potrykus, I. [1991] Ann. Rev. Plant Physiol. Plant Molec. Biol. 42:205-225; Vasil, V., A. M. Castillo, M. E. Fromm, I. K. Vasil [1992] Bio/Technology 10:667-674). Examples of some of these methods, as outlined below, illustrate the breadth of techniques which have been attempted for producing transgenic cereals but have been unsuccessfully applied to wheat.
Agrobacterium and Cereals
The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens has been shown to introduce foreign genetic information into plant cells. International Publication No. WO84/02920. However, A. tumefaciens is incapable of infecting monocotyledons, such as cereals. Therefore, it does not transfer DNA to these types of plants, such as wheat. The recent report of transgenic wheat obtained by using Ti from Agrobacterium (Hess, D., K. Dressier, R. Nimmrichter [1990] Plant Sci. 72:233-244), can be considered artifactual based on criteria used to demonstrate transformation (Potrykus, I. [1991], supra; Potrykus, I. [1990] Bio/Technology 8:535-542). Raineri et al. (Raineri, D. M., P. Bottino, M. -P. Gordon, E. W. Nester [1990] Bio/Tech. 8:33-38) presented evidence giving a reasonable inference of Agrobacterium-mediated transformation of rice cells (Oryza sativa), although definitive proof is still lacking.
Agroinfection and Cereals
Agroinfection has been shown to lead to systemic spread of maize streak virus. This showed for the first time that Agrobacterium can transfer its T-DNA to cereal cells. Later it was demonstrated that the efficiency of transfer is comparable to dicot systems. In cereals, agroinfection leads to the transfer of the virus-carrying T-DNA into wound-adjacent cells. The virus is released, replicates, and spreads systemically. Even if it reaches rare competent cells somewhere in the plant body it does not integrate. Thus the chances that agroinfection will produce transgenic cereals are minimal and no different from normal Agrobacterium infection.
Viral Vectors
Viruses spread systemically throughout the plant from the infection site and can replicate many thousands of copies per cell. According to the available evidence, viruses do not integrate into the host genome and they are excluded from meristems and thus from transmission to sexual offspring.
Incubation in DNA of Dry Seeds or Embryos
Thus far, no transgenic plants have been recovered using this method. Incubation of seeds in DNA has yielded indicative evidence since the 1960s. However, no proof for transformation has ever been presented. Although some experiments clearly demonstrate the presence and expression of defined marker genes as well as the replication of engineered viral DNA, they do not provide proof for transformation. The conclusion that the data demonstrate uptake of the foreign DNA into the cells of the embryos is merely one hypothesis. It is not possible to exclude the alternative hypothesis that the DNA data are the result of open cells at the large wound site and that the virus DNA data are, in addition, due to systemic spread.
Incubation in DNA of Tissues or Cells
No transgenic tissues or plants have been recovered. There have been many approaches where seedlings, organs, tissues, cells, or cell cultures of numerous plant species have been brought into direct contact with foreign DNA and defined marker genes. Even in experiments that would have recovered extremely rare events of integrative transformation, there is not a single proven case of integrative transformation.
Pollentube Pathway
No transgenic plants have been recovered from this method. Integration into high molecular weight DNA and defined hybrid fragments has not been shown.
Liposome Fusion with Tissues and Protoplasts
Fusion of DNA-containing liposomes with protoplasts is an established method for the production of transgenic plants. DNA-containing liposomes have also been applied to various tissues, cell cultures and pollen tubes, with the rationale that liposomes might help transport the DNA via plasmodesmata or directly across the cell wall. It has been shown that liposomes can carry small dye molecules into cells within tissues via fusion with the plasmalemma. There is, however, no proof for transport and integration of marker genes. As plasmodesmata are sealed off immediately upon wounding, this route is not open even for small liposomes. Integration of the cell wall with phospholipids does not seem to change its barrier function. No transgenic cereals have been obtained by this method.
Liposome Injection
Thus far no transgenic tissue has been recovered by this method. Microinjection of DNA has yielded transgenic chimeras. Microinjection into differentiated cells can easily deposit the DNA into the vacuole, where it is degraded. Microinjection of liposomes into the vacuole leads to fusion with the tonoplast thus releasing the content of the liposome into the cytoplasm, as demonstrated with cytoplasm-activated fluorescent dyes.
Protoplasts and Direct Gene Transfer
Protoplasts efficiently take up DNA if treated with polyethylene glycol (PEG) and/or electroporation. When protoplasts are transformed that are also competent for regeneration, transgenic plants can be recovered that stably contain, express, and inherit the foreign gene. Protoplasts isolated from intact cereal plants do not contain cells competent for regeneration. Competent protoplasts have, so far, been isolated only from embryogenic suspensions established from immature tissues (scutellum, leaf base, anther). Standard direct gene transfer procedures with protoplasts from embryogenic suspensions has led to the regeneration of transgenic rice (Oryza sativa var. japonica and indica) and maize (Zea mays). However, the establishment of the appropriate cell cultures is an art that depends upon parameters which are difficult to control.
Protoplasts from Cereal Plants
No transgenic clones have been recovered by this method. As the establishment of appropriate embryogenic suspensions is a delicate and often unpredictable process, it would be of great advantage if protoplasts isolated directly from differentiated tissues could be cultured. However, this approach appears, to date, rather hopeless because differentiated cereal tissues do not express the wound response and do not contain cells competent for regeneration. DNA uptake is no problem, as can be shown easily with transient expression assays. If integration occurs it has no consequences, because protoplasts do not proliferate.
Biolistics or Particle Gun
Acceleration of heavy particles covered with DNA has been used with some success to transport genes into plant cells and tissues. Transgenic plants have been produced in soybean and tobacco. The method has the potential for general applicability: (1) it is easy to handle; (2) one shot can lead to multiple hits; (3) cells survive the intrusion of particles; (4) the genes coated onto the particle resume biological activity; (5) target cells can be as different as pollen, cell cultures, plant organs, and meristems; (6) particles also reach deeper cell layers. Thus, the method provides a biological vector-independent DNA delivery system. Transgenic maize plants have been obtained by the delivery of DNA-coated particles into plated suspension cultures (Fromm, M. E., F. Morrish, C. Armstrong, R. Williams, J. Thomas, T. M. Klein [1990] Bio/Technology 8:833-839; Gordon-Kamm, W. J., T. M. Spencer, M. L. Mangano, T. R. Adams, R. J. Daines, W. G. Start, J. V. O'Brien, S. A. Chambers, W. R. Adams Jr., N. G. Willetts, T. B. Rice, C. J. Mackey, R. W. Krueger, A. P. Kausch, and P. G. Lemaux [1990] The Plant Cell 2:603-618). Bombardment of intact embryos, pollen and other tissues has not yielded any transgenic wheat.
Microinjection
No transgenic offspring have been recovered so far in cereals using this method. Microinjection uses microcapillaries and microscopic devices to deliver DNA into defined cells in such a way that the injected cell survives and can proliferate. This technique has produced transgenic clones from protoplasts (where transformation via direct gene transfer is easier) and transgenic chimeras from microspore-derived proembryos in oilseed rape. As with biolistics, microinjection definitely delivers DNA into cells. Extensive experiments with many species, including cereals, have not definitively produced any transgenic plants.
Pollen Transformation
No transgenic plants have been produced by this method. This approach is based on the hope that DNA could be taken up into germinating pollen and either integrate into the sperm nucleus or reach the zygote with the pollen tube. Numerous large-scale experiments in experienced laboratories with defined marker genes have only given clearly negative results. Therefore, it is justified to conclude that this approach is not a very promising one.
Electroporation
No transgenic clones have been produced when applied to cells and tissues. Not much potential is expected with walled cells. This is a routine method for gene transfer to protoplasts but transfer of genes into walled cells has not been possible.
In summary, the two methods found useful for delivery of DNA into cells of cereal plants which result in stable transformation are electroporation or polyethylene glycol treatment into protoplasts, and more recently, accelerated particle bombardment into plated suspension cultures or calli. Attempts at producing transgenic wheat plants, however, proved unsuccessful, although methods were utilized that were previously successfully applied to the regeneration of other plants, including other cereals.
Following is a summary of the methods attempted to obtain stably transformed callus tissues and plants of wheat. These methods, though previously shown to transform cereal plants such as rice and maize, failed to result in the production of viable wheat plants.
1. Protoplasts
Plasmid DNA containing NPTII and GUS genes was introduced into embryogenic protoplasts by the PEG method. A number of cell colonies selected on kanamycin media tested positive for GUS activity. Plants were obtained from these colonies. However, Southern analyses provided no evidence for the presence of either the NPTII or the GUS gene. It was therefore concluded that the available protoplast system was not suitable for obtaining transgenic plants of wheat.
2. Suspension Culture
Next, plasmid DNA containing NPTII, GUS, and EPSPS genes was delivered into intact plated suspension culture cells by bombardment with accelerated microprojectiles (Vasil, V., S. M. Brown, D. Re, M. E. Fromm, I.K. Vasil [1991] Bio/Technology 9:743-747). Calli resistant to kanamycin and showing GUS expression were shown by Southern analyses to have integrated each of the three genes. Expression of the genes was demonstrated by NPTII and EPSPS activity. No transgenic plants were recovered from the transformed calli.
3. Immature Embryos
Next, immature zygotic embryos of wheat were bombarded with DNA-coated macroprojectiles at various intervals after excision and culture. Transient expression of the GUS gene was demonstrated, but no stably transformed callus lines or plants were obtained.
Thus, the methods known in the art to show the greatest potential for producing transgenic plants did not viably transform wheat plants, even though success had been shown in rice and maize.